Cytotoxicity comparison of 35 developmental neurotoxicants in human induced pluripotent stem cells (iPSC), iPSC-derived neural progenitor cells, and transformed cell lines

Highlights

• Developmental neurotoxicity (DNT) of 35 chemicals is examined by MTS/ATP assays.

• iPSC and NPC are more vulnerable to most DNT chemicals than COS-7 and HepG2 cells.

• 14 DNT chemicals induce differential inhibitions of iPSC differentiation to NPC.

• RT-qPCR of (un)differentiation marker genes is useful for in vitro DNT evaluation.

Abstract

The Organization for Economic Co-operation and Development (OECD) test guideline 426 for developmental neurotoxicity (DNT) of industrial/environmental chemicals depends primarily on animal experimentation. This requirement raises various critical issues, such as high cost, long duration, the sacrifice of large numbers of animals, and interspecies differences. This study demonstrates an alternative protocol that is simple, quick, less expensive, and standardized to evaluate DNT of many chemicals using human induced pluripotent stem cells (iPSC) and their differentiation to neural progenitor cells (NPC). Initially, concentration-dependent cytotoxicity of 35 DNT chemicals, including industrial materials, insecticides, and clinical drugs, were compared among iPSC, NPC, and two transformed cells, Cos-7 and HepG2, using tetrazolium dye (MTS)-reducing colorimetric and ATP luciferase assays, and IC₅₀ values were calculated. Next, inhibitory effects of the 14 representative chemicals (mainly insecticides) on iPSC differentiation to NPC were evaluated by measuring altered expression of neural differentiation and undifferentiation marker genes. Results show that both iPSC and NPC were much more sensitive to most DNT chemicals than the transformed cells, and 14 chemicals induced differential patterns of marker gene expression, highlighting the validity and utility of the protocol for evaluation and classification of DNT chemicals and preclinical DNT tests for safety assessment.

Introduction

Epidemiological studies suggest close association between embryonic/postnatal exposure to some industrial chemicals and the onset of neurobehavioral disorders, including learning disabilities, attention deficit hyperactivity disorder (ADHD), autism, and the other cognitive abnormalities, in millions of children worldwide (Landrigan et al., 2012; Grandjean and Landrigan, 2014; Ross et al., 2015). These chemicals include insecticides/fungicides, industrial solvents, catalysts/plasticizers, clinical drugs, and research reagents (Pei et al., 2016; Harrill et al., 2018). Some of these chemicals have already been banned. The central nervous system in the fetal and neonatal periods is especially vulnerable to such chemicals, perhaps because the blood-brain barrier is not yet complete (Tohyama, 2016) when critical processes of temporal/regional neural development are ongoing (Rice and Barone Jr., 2000). Neurobehavioral disorders affect ~10% of all newborns/children, and prevalence of ADHD in the US young (3–17 years) population increased from 7.2% (in 2007) to 8.5% (in 2011) (Bloom et al., 2009; Bloom et al., 2012). Genetic factors play substantial roles–perhaps 30%–40% of all neurobehavioral disorders are due to genetics, but non-genetic environmental factors, including chemical exposure, are also involved (Grandjean and Landrigan, 2014).

To date, DNT behavioral/neurological test methodologies depend heavily on experimental animals, mainly rats (TG426; Developmental Neurotoxicity Study). Significant limitations with animal experimentation under this guideline are high cost, long duration, the sacrifice of large numbers of animals, interspecies differences, and lack of skilled laboratory animal technicians in the face of increasing demands (Schmidt, 2009; Tsuji and Crofton, 2012; Tohyama, 2016; Taylor, 2018). The Organization for Economic Co-operation and Development (OECD) has begun discussion on a DNT in vitro guidance document for protection of developing brains from chemicals that cause DNT (Fritsche et al., 2018b; Sachana et al., 2019). The basic concept is that the complex procedure of brain development can be disassembled into several neurodevelopmental endpoints which can be represented by a combination of different alternative assays (Fritsche et al., 2018a). The discovery of induced pluripotent stem cells (iPSC) and their differentiation to various cell lineages provides an opportunity for application to DNT evaluation (Pei et al., 2016; Ryan et al., 2016; Bal-Price et al., 2018b; Barenys and Fritsche, 2018; Fritsche et al., 2018a). iPSC are not tumor cells but proliferate infinitely and they can differentiate to neural cell lineages.

We assume that DNT in early stage of neural differentiation consists of two components: cytotoxicity to neural cells and differentiation alteration activity on neural stem/progenitor cells; therefore, we initially compared concentration-dependent cytotoxic effects of DNT chemicals selected by National Toxicity Program among iPSC, neural progenitor cells (NPC), and two transformed cell lines. Subsequently, we examined the impacts of 14 representative DNT chemicals on iPSC differentiation to NPC. These results support the utility of iPSC/NPC to supplement animal experimentation for the evaluation of DNT in safety assessment.